The two-faced neo-epitope: innovative immunoinformatic approaches for mutanome-directed Leonard Moise1,2, Guilhem Richard1, Frances Terry1, William Martin1, Anne S. De Groot1,2 1 EpiVax, Inc., Providence, RI, United States; 2 Institute for Immunology and Informatics, University of Rhode Island, Providence, RI, United States Abstract Mutanome-Directed Cancer Immunotherapy JanusMatrix Discovers Potential Treg Neo-Epitopes Introduction: Optimal epitope selection is a significant Tumor Biopsy Expressed Discovery Neo- Discovery Immunotherapy JanusMatrix separates the amino acid sequence of epitopes into TCR-facing residues (epitope) and challenge in designing effective personalized vaccines Tumor for cancer immunotherapy. Although epitope predictors Mutanome cell HLA binding cleft-facing residues (agretope), then compares the TCR face to other putative T cell epitopes.

rapidly identify candidates, a large proportion prove to be + IMMUNOINFORMATICS CD8 non-immunogenic for reasons that are not well T cell Cross-reactive peptides: understood. • Are predicted to bind the same MHC allele. Neo-epitopes • Share same/similar T cell-facing residues.

Methods: We use the EpiMatrix and JanusMatrix Normal Tumor CD4+ TCR cross-reactivity prediction: algorithms, previously validated in prospective vaccine T cell APC • Given a protein or peptide, T cell epitopes are identified based on MHC contacts using studies for infectious diseases, to identify patient-specific EpiMatrix. sequences displaying TCR-facing residues with • JanusMatrix searches for potentially cross-reactive TCR by screening TCR-facing residues increased potential to induce T effectors and reduced Ancer Platform: the “Answer” to Cancer against a preloaded, EpiMatrix-processed reference database. • Peptides with high cross-reactive potential are associated with reduced INFγ secretion in potential to induce thymic-educated Tregs. PBMCs of healthy donors (Liu et al. Human Vaccines & Immunotherapeutics, 2015). Scan for HLA Matched Epitopes Results: Our tools selected immunogenic neo-epitopes (Patient Class I + II) with EpiMatrix with 72% accuracy, compared to 21% when using Human IFNγ responses to cross-conserved peptides identified by JanusMatrix From (figures adapted from Liu R et al. Human Vaccines & Immunotherapeutics, 2015) publicly available tools, in an analysis of mutanome- mutanome derived peptides [Strønen 2016]. Analysis of cancer to vaccine Screen for Humanness and Identify vaccine efficacy studies performed in mice [Kreiter 2015] “Non-Self” Neo-epitopes with JanusMatrix also show that vaccines effective at preventing tumor design in growth contain higher numbers of neo-epitopes with 1-2 weeks lower self-cross-reactive potential. Rank and Select Peptide Candidates Immune Engineering of vaccines with Conclusions: Analyzing both faces of the T cell epitope JanusMatrix. Treg epitopes can be identifies higher value epitopes than conventional identified in and effectively approaches for more immunogenic personalized Design: Commercial-grade Advanced removed to increase the immunogenicity Peptide pool with low Peptide pool with high Peptide pool Peptide pool of vaccines. vaccines. Vaccine Design with VaxCAD JanusMatrix scores JanusMatrix scores + + not similar to human similar to human Ø JanusMatrix-derived Treg epitope Retrospective Immunoinformatic Analyses of Published Cancer Datasets Ancer Analysis of Mutated Peptides from Strønen et al. Science 2016 Ancer Analysis of Cancer Vaccines from Kreiter et al. Nature 2015

• Summary of the Strønen et al. Science 2016 study: • Summary of the Kreiter et al. Nature 2015 study: HLA-A2-restricted mutated peptides derived from melanoma patients were identified using NetMHC and NetMHCpan. Few peptides Pentatopes were constructed from mutated sequences derived from colon carcinoma model CT26. Pentatope 2 showed greater antitumor (21%) selected with standard in silico tools were immunogenic when tested in T cell assays using PBMCs of healthy donors and activity compared to Pentatope 1 in BALB/c mice inoculated with CT26 tumor cells (right). patients’ TILs. Experimentally determined peptide-MHC off-rates can be used to discriminate immunogenic from non-immunogenic • Pentatope 2 increased anti-tumor activity may be explained by a higher number of Class I and II neo-epitopes and overall lower peptides with 65% accuracy. potential for cross-reactivity, as identified by Ancer (left). • Immunogenic peptides have significantly greater binding potentials than non-immunogenic peptides, as measured by EpiMatrix (Mann-Whitney test, p = 0.0020). No significant difference is observed between peptides when evaluated with publicly Class I Neo-epitope content Class II Neo-epitope content available in silico tools (Mann-Whitney test, p = 0.0561). 3 2 • Ancer can differentiate immunogenic and non-immunogenic peptides with 72% accuracy by using strict neo-epitope definitions and by evaluating their potential for cross-reactivity. PPV of Ancer is twice the one of standard in silico tools. Adapted from Extended Data Figure 3b. 2 Kreiter et al., Nature 520, 692–696. 2015 ) Predicted binding affinities of mutated peptides Accuracy, PPV, and speed of neo- 1 epitopes

epitope predictors - Ancer ( 1 Neo epitope per sequence per epitope sequence per epitope - - 0.72 0.42 Neo Neo 0.65 0 0 0.38 Pentatope 1 Pentatope 2 CT26-M19 Pentatope 1 Pentatope 2 CT26-M19

Class I Murine cross-reactivity Class II Murine cross-reactivity potential potential 0.21 4 4

0.21 3 3 ) Predicted Binding Predicted Binding

in silico in vitro in silico Ancer 2 2 minutes months minutes ( reactivity reactivity potential - 1 1 Murine Janus Score Janus Murine Score Janus Murine Cross 0 0 Pentatope 1 Pentatope 2 CT26-M19 Pentatope 1 Pentatope 2 CT26-M19 Conclusions Sequence analysis of the MHC- and TCR-facing residues of T cell epitopes enables prediction of epitope phenotype. Epitopes that share a TCR-face with numerous human sequences may activate Tregs. Sharper definition of neo-antigens by immunoinformatic analysis may improve epitope selection for mutanome-directed cancer immunotherapy. In silico analyses of cancer vaccines suggest that high neo-epitope density and low cross-reactivity potential may be indicative of improved clinical outcomes. References

• Moise L, Gutierrez A, Kibria F, Martin R, Tassone R, Liu R, Terry F, Martin B, De Groot AS. iVAX: An integrated toolkit for the selection and optimization of antigens and the design of epitope-driven vaccines. Hum Vaccin Immunother. 2015;11(9):2312-21. • Moise L, Gutierrez AH, Bailey-Kellogg C, Terry F, Leng Q, Abdel Hady KM, VerBerkmoes NC, Sztein MB, Losikoff PT, Martin WD, Rothman AL, De Groot AS. The two-faced T cell epitope: examining the host-microbe interface with JanusMatrix. Hum Vaccin Immunother. 2013 Jul;9(7):1577-86. • Liu R, Moise L, Tassone R, Gutierrez AH, Terry FE, Sangare K, Ardito MT, Martin WD, De Groot AS. H7N9 T-cell epitopes that mimic human sequences are less immunogenic and may induce Treg-mediated tolerance, Human Vaccines & Immunotherapeutics. 2015 11:9, 2241-2252 • Wada Y, Nithichanon A, Nobusawa E, Moise L, Martin WD, Yamamoto N, Terahara K, Hagiwara H, Odagiri T, Tashiro M, Lertmemongkolchai G, Takeyama H, De Groot AS, Ato M, Takahashi Y. A humanized mouse model identifies key amino acids for low immunogenicity of H7N9 vaccines. Sci Rep. 2017 Apr 28;7(1):1283 • Kreiter S, Vormehr M, van de Roemer N, Diken M, Löwer M, Diekmann J, Boegel S, Schrörs B, Vascotto F, Castle JC, Tadmor AD, Schoenberger SP, Huber C, Türeci Ö, Sahin U. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature. 2015 Apr 30;520(7549):692-6. • Strønen E, Toebes M, Kelderman S, van Buuren MM, Yang W, van Rooij N, Donia M, Böschen ML, Lund-Johansen F, Olweus J, Schumacher TN. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science. 2016; 352(6291), 1337-41. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee W, Yuan J, Wong P, Ho TS, Miller ML, Rekhtman N, Moreira AL, Ibrahim F, Bruggeman C, Gasmi B, Zappasodi R, Maeda Y, Sander C, Garon EB, Merghoub T, Wolchok JD, Schumacher TN, Chan TA. Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer. Science. 2015 Apr;348(124):124-8

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